This invention relates to the stabilization of fluorescent conjugates, in particular to stabilization of biological reagents, such as proteins, peptides, ligand analogues and nucleic acids, conjugated to a fluorescent molecule or to a fluorescent particle.
In order to determine the presence or concentration of target ligands or analytes in a fluid, immunoassays utilize binding proteins, such as antibodies, to specifically bind to target ligands or analytes. Commonly, a signal is generated from the immunoassay to provide a detectable or sensible result. A class of useful signal generators are fluorescent molecules because of their high specific activity.
Fluorescent molecules have been utilized in immunoassays as signal generators in several ways. For example, enzymes can catalyze the production of fluorescent dyes from a substrate, such that the amount of fluorescent dye generated is related to the presence or amount of analyte or target ligand in a sample; such enzymes can be conjugated to antibodies or ligand analogues. Fluorescent dyes can also be conjugated to antibodies or ligand analogues, and the amount of fluorescent signal is measured as a result of the assay process to define the analyte presence or amount.
An objective with immunoassays is to make them more sensitive, so that a lower concentration of analyte can be measured. To increase sensitivity, one can conjugate a biological reagent to a fluorescent particle. As used herein, xe2x80x9cfluorescent particlesxe2x80x9d can be formed of a synthetic material such as latex, can comprise a natural material such as liposomes, or combinations of natural and synthetic materials. In the preparation of the fluorescent particles, the fluorescent dyes or enzymes are generally imbibed into and/or adsorbed onto the particles, using mixed organic and aqueous solutions. The fluorescent dyes or enzymes incorporated into particles are usually water insoluble, whereas the dyes or enzymes conjugated directly to biological reagents are generally water soluble.
One can conjugate, for example, a biological reagent to a fluorescent particle that contains many fluorescent molecules or enzymes capable of converting a substrate into a fluorescent product. In the case of conjugating an antibody to a fluorescent particle, the analyte that is bound by the antibody conjugated to the fluorescent particle containing many fluorescent molecules is thus associated with a larger signal as compared with binding an antibody that is conjugated with a single fluorescent molecule or enzyme.
Examples of immunoassays that incorporate fluorescent dyes or enzymes are numerous and some are described in U.S. Pat. Nos. 4,283,382; 4,351,760; 4,420,568; 4,868,132; 4,876,190; 5,075,215; and, E.P.O. Application Number PCT/US87/03226.
Immunoassays are characterized as either competitive or non-competitive. Competitive immunoassays generally operate by a competition between a ligand analogue signal generator complex and a ligand (i.e., the analyte) for binding to a limited amount of antibody. Competitive immunoassays can also function by a competition between a protein or an antibody signal generator conjugate and an analyte which is a protein or an antibody, respectively, for binding to a limited amount of ligand. Non-competitive immunoassays generally function by having two different antibodies bind the analyte at different epitopes, one antibody conjugated to the signal generator and the other antibody in solution or bound to a solid phase in order to facilitate separation of unbound signal generator from the bound signal generator.
The antibody concentration in an immunoassay generally dictates the kinetics of the immunoassay, as well as the fraction of analyte bound to the antibody. In general, to accelerate the binding of an analyte to an antibody so that the reaction achieves a desired reaction rate or equilibrium in a desired time, the highest antibody concentration that is feasible is used in the assay; this is particularly true for non-competitive immunoassays. Thus, a high antibody concentration is often employed in fluorescent assays. Generally a high antibody concentration is also associated with a high fluorescent dye concentration, since each make up the antibody:particle conjugate or the antibody:dye conjugate.
Thus, for immunoassays, fluorescent particles are often used to obtain a more sensitive measure of analyte concentration. The quantity of a fluorescent molecule that needs to be associated with a complex of an analyte and a fluorescent conjugate is generally determined based on the sensitivity requirements of the assay. Thus, for sensitive assays, the amount of fluorescent dye or enzyme associated with the analyte in the assay process, is maximized. In such a case, the local concentration of fluorescent molecules is very high in the immediate vicinity of the biological reagent because they are in close proximity to each other.
Nucleic acid assays also utilize fluorescent signal generators. Signal generators associated with nucleic acids are used in assays to measure nucleic acids complementary with the nucleic acid associated with a signal generator. In order to optimize the hybridization kinetics with the target nucleic acid and also to achieve the most sensitive assay, these nucleic acids are also at a high local concentration in the vicinity of fluorescent molecules. To create a nucleic acid conjugate, a signal development element is conjugated to a nucleic acid sequence. The signal development element can be a fluorescent particle or a fluorescent molecule.
Previously, however, a problem was encountered with fluorescent conjugates for use in assays. Often, fluorescent conjugates were not stable. The reactivity of such conjugates was found to decay over time, such that the function or properties of a biological reagent in a fluorescent conjugate would change with time. For example, the binding affinity of an antibody linked to a fluorescent signal would often decrease with time. The mechanism(s) for this degradation of the properties of biological reagents in fluorescent conjugates was not known. It was presumed, however, that the degradation occurred consequent to the release or uncoupling of the biological reagent from the fluorescent molecule.
For the first time in the art, the mechanisms leading to the degradation of biological reagents in fluorescent conjugates have been identified. Accordingly, compositions and methods are provided to modulate this degradation, both in fluorescent conjugates and in the reagents used to synthesize the fluorescent conjugates. The effectiveness of fluorescent conjugates for use in assays is enhanced pursuant to the present invention.
Thus, disclosed is a fluorescent conjugate comprising a biological reagent, a fluorescent molecule, and a means for impeding phototoxic degradation of the biological reagent. The impeding means can comprise a cross-linking substance having a long molecular distance, whereby the cross-linking substance links the fluorescent molecule and the biological reagent; a protein; a quencher of singlet oxygen; a quencher of a free radical; a system for depleting oxygen; or a combination thereof.
The conjugate can comprise that the fluorescent molecule is directly linked to the biological reagent. The conjugate can further comprise a particle which comprises a fluorescent molecule; the particle can comprise a natural material or a synthetic material. For example, the particle can comprise a natural material which is alumina, silica, or, a liposome. The particle can further comprise an oxygen depleting system.
Disclosed is a solution comprising a fluorescent conjugate and an oxygen depleting system. For an embodiment of the fluorescent conjugate to be placed in a solution, the solution can comprise a means for impeding phototoxic degradation. The impeding means of the solution can comprise an antioxidant, a protein, an oxygen depleting system, or a combination thereof. The fluorescent conjugate of the solution can comprise a biological reagent, a fluorescent molecule, and a means for impeding phototoxic degradation of the biological reagent. The impeding means of the conjugate in the solution can comprise: a system to deplete oxygen; a cross-linking substance having a long molecular distance, whereby the cross-linking substance links the fluorescent molecule and the biological reagent; an antioxidant; a protein; or, a combination thereof.
Also disclosed is a fluorescent conjugate prepared by a process comprising steps of: providing a biological reagent; providing a fluorescent molecule; providing a heterofunctional linking reagent; chemically reacting the biological reagent, the fluorescent molecule, and the heterofunctional linking reagent, whereby the biological reagent becomes linked to the fluorescent molecule; and; associating an oxygen depleting system, a protein, an antioxidant, or a combination thereof, with the biological reagent or the fluorescent molecule prior to the reacting step; or, associating an oxygen depleting system, a protein, an antioxidant, or a combination thereof, with the linked biological reagent and fluorescent molecule following the reacting step. The heterofunctional linking reagent can be: a heterofunctional polyethylene glycol (PEG) derivative, a long chain form of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), a long chain form of succinimidyl 3-(2-pyridyldithio)propionate (SPDP), or, a heterofunctional peptide derivative.
Disclosed is a method of use of an antioxidant, a protein, an oxygen depleting system, or a cross linking substance to impair the phototoxic degradation of a biological reagent in a fluorescent conjugate comprising the biological reagent and a fluorescent molecule, or to impair the phototoxic degradation of the material used to prepare a composition comprising a fluorescent molecule, the method occurring by combining an antioxidant, a protein, an oxygen depleting system, or a cross linking substance with the fluorescent conjugate, or with the material used to prepare a composition comprising a fluorescent molecule.
Also disclosed is a method to facilitate preparation of a stable fluorescent composition, where the composition comprises a fluorescent molecule which absorbs light and provides a fluorescent result upon exposure to light of a defined spectrum. The method comprises: providing an environment comprising diminished amounts of light from the defined spectrum, relative to a white light environment; providing the fluorescent molecule, or a material to prepare the composition; placing the fluorescent molecule or the material to prepare the composition into the provided environment. An embodiment of the method is disclosed which comprises: providing the fluorescent molecule; providing the material to prepare the composition; placing the fluorescent molecule into the provided environment; placing the material to prepare the composition into the provided environment; and, combining the fluorescent molecule and the material to prepare the composition; whereby phototoxic degradation of the material to prepare the composition is diminished upon completion of the method. When this embodiment of the method is performed, the combining step can occur before the step of placing the fluorescent molecule into the provided environment and before the step of placing the material to prepare the composition into the provided environment.
Fluorescent moleculexe2x80x94As used herein, a fluorescent molecule is one that either emits light during the transition from an excited electronic state to a lower energy state, or is an enzyme or catalyst that is capable of acting on a substrate to yield a molecule that emits light during the transition from an excited electronic state to a lower energy state.
Fluorescent dyesxe2x80x94Fluorescent dyes are those that emit light during the transition from an excited electronic state to a lower energy state. The lower energy state is commonly referred to as the ground state. The excited state is attained by absorption of light at an appropriate wavelength. One pathway for relaxation of the excited state is the emission of light. Fluorescent dyes exhibit singlet-singlet emission of light.
Fluorescent particlesxe2x80x94A particle which comprises one or more fluorescent molecules. A fluorescent particle can be formed of a synthetic material such as latex, can comprise a natural material such as liposomes, or can comprise a combination of natural and synthetic materials.
Ligandxe2x80x94A binding partner to a ligand receptor or to a complementary nucleic acid sequence.
Ligand analoguexe2x80x94A chemical derivative of a ligand. A ligand analogue may be attached either covalently or noncovalently to another species, for example, to a signal development element. Ligand analogue and ligand may be the same and both are capable of binding to ligand receptor.
Ligand Receptorxe2x80x94A receptor capable of binding ligand. Typically, a ligand receptor is an antibody or a complementary nucleic acid sequence, but may also be a ligand.
Signal Development Elementxe2x80x94The element of the biological reagent conjugate that produces a detectable signal. The signal can be directly produced by the signal development element, or produced upon exposure of the signal development to a substrate.
Radicalxe2x80x94A radical is an atom or a group of atoms that possess an unpaired electron. Radicals are also referred to as free radicals, diradicals and radical pairs.
Biological Reagentxe2x80x94A moiety, e.g., a protein, peptide, ligand analogue, or nucleic acid capable of being associated with a signal development element in a conjugate.
Fluorescent conjugatesxe2x80x94A fluorescent conjugate comprises a biological reagent, e.g., a protein, peptide, ligand analogue or nucleic acid sequence, in association with a fluorescent molecule, a fluorescent particle, an enzyme, or a generator of fluorescent dyes. The association can be covalent or non-covalent.
The fluorescent molecules can be conjugated with natural or synthetic particles to form fluorescent particles. For example, a fluorescent molecule can be adsorbed to alumina or silica particles, or imbibed into latex particles or liposomes; the proteins, peptides, ligand analogues or nucleic acids can be adsorbed or covalently coupled to the particles to make the fluorescent conjugates. Alternatively, fluorescent molecules can be directly coupled to the proteins, peptides, ligand analogues or nucleic acids to form fluorescent conjugates. The direct coupling can be a covalent bond. The fluorescent molecules utilized for these latter conjugates are generally water soluble having a carboxylic acid, thiol, amine, maleimide, alkyl halide or N-hydroxy succinic ester moiety to facilitate coupling to the proteins, peptides, ligand analogues or nucleic acids.
Antioxidantxe2x80x94A substance such as a quencher which, when present in a system significantly delays or inhibits oxidation of the substrate.
Reactive speciesxe2x80x94Examples of reactive species are singlet oxygen, free radicals and excited triplet states.
Quencherxe2x80x94A substance which either through physical means, such as energy transfer, or through chemical means delays or inhibits the decomposition of another substance.
For the first time in the art, the mechanisms that lead to the degradation of biological reagents in fluorescent conjugates have been identified. It has been found that ambient white light leads to the generation of singlet oxygen or radicals which act on biological reagents. The studies that led to this finding are disclosed, as are compositions and methods to modulate this degradation. Pursuant to the present invention, the effectiveness of fluorescent conjugates for use in assays is enhanced.
Although the degradation of biological reagents in fluorescent systems had been noted, the causative mechanism was unknown. Surprisingly, it was found that biological reagents such as antibodies, peptides and ligand analogues, as well as reagents used to synthesize the fluorescent conjugates, are degraded by a mechanism that is induced upon exposure of these materials to routine ambient conditions. The ambient conditions can constitute mere exposure to white light and oxygen.
Accordingly, this invention concerns the stabilization of proteins, peptides, ligand analogues and nucleic acids that are comprised in fluorescent conjugates, as well as stabilization of the materials to make fluorescent conjugates.
The inactivation of antibody binding activity in immunoassays or inactivation of nucleic acid hybridization assays by singlet oxygen or radicals has not been previously described. Previously, only the inactivation of lysozyme and the oxidation of amino acids have been described (Singlet Oxygen, 1985, Vol. IV, p. 91-143, ed. A. A. Frimer, CRC Press, Boca Raton, Fla.; Int. J. Radiat. Biol. (1986) 50:41-45); however, these descriptions are related to fluorescent dyes that are not conjugated to proteins, peptides, ligand analogues and nucleic acids; unlike the present invention, these dyes and biological reagents were freely soluble in aqueous solutions.
Furthermore, it has been noted that phthalocyanines can also initiate phototoxicity by an electron transfer mechanism (Type 1) in which the fluorescent dye, in an excited triplet state, can undergo reactions with neighboring molecules by an electron or hydrogen transfer process. The production of singlet oxygen (Type II) by the excitation of fluorescent dyes has been utilized for photodynamic therapy (Polym. Adv. Technol. (1995) 6:118-130; Int. J. Radiat. Biol. (1995) 67:85-91; Ciba Foundation Symposium (1989) 146:17-26) and in immunoassays, whereby singlet oxygen generation by particles comprised of silicon phthalocyanine has been used to generate a delayed luminescence. The delayed luminescence is proportional to the analyte concentration (Clin. Chem. 42:1518-1526 (1996)).
The teachings herein show that light (ambient or focused) decreased the effectiveness in binding reactions of antibodies, peptide and ligand analogue fluorescent conjugates. The mechanism for the decrease in effectiveness is believed to be related to the generation of reactive species, such as singlet oxygen or radicals, by the fluorescent molecules. The inactivation of biological reagents in fluorescent conjugates makes them unreliable for use in assays. The inactivation of antibody binding in immunoassays or of nucleic acid hybridization in nucleic acid assays is generally manifest by a decrease in the slope of a dose response curve of the assay.
As disclosed herein, light-mediated inactivation of biological reagents in fluorescent conjugates is very dramatic. It has been found that the degradation of biological reagents in fluorescent conjugates is independent of the fluorescent molecule used in making the fluorescent conjugate. The efficiency of the inactivation is believed to be a result of two factors: First, the high local concentrations of both the dye and the biological reagent; and, second, the relative proximity of the biological reagent and the fluorescent dye in the fluorescent conjugates.
The presumed mode of degradation is type I and/or type II mechanisms of phototoxicity. In the case of type I mechanisms, the triplet sensitizer, that is, the triplet state of a fluorescent dye, reacts with nearby molecules through an electron or hydrogen transfer process. Free radicals are thus formed; namely the semireduced sensitizer and the semioxidized substrate. The semireduced sensitizer can react with oxygen to form a superoxide radical anion, which can further react with other molecules. The semioxidized sensitizer can become reduced by another molecule to form a semioxidized substrate, which can further react.
In the case of type II mechanisms, the triplet sensitizer can react with ground state oxygen to form singlet oxygen which can further react with other molecules, such as biomolecules and reagents to give oxidized products. (See, e.g., Int. J. Radiat. Biol. (1995) 67:85-91).
As disclosed herein, biological reagents associated with fluorescent dyes become inactivated or ineffective through the triplet state of the dye. The generation of triplet states of fluorescent dyes can result in the formation of a variety of radical species, including but not limited to singlet oxygen, superoxide radicals, hydroxyl radicals and organic radicals. These radical species have been known to cause damage to biological cells and proteins. Accordingly, these reactive triplet state species react with the surface of the biological reagent. Consequently, there will be a decreased effectiveness of an antibody for binding to analyte, or for a peptide or ligand analogue for binding to antibody. The inventive teachings set forth compositions which slow or prevent the light-induced destruction of biological reagents.
This invention relates to the stabilization of biological reagents conjugated to fluorescent particles or conjugated to a fluorescent molecule, as well to the preparation of such conjugates. The biological reagent can comprise a protein, peptide, ligand analogue or nucleic acid component. When a biological reagent is stabilized in accordance with the invention, phototoxic degradation of the biological reagent in a fluorescent conjugate is impeded.
Accordingly, it has now been found that inactivation of biological reagents conjugated to fluorescent molecules or fluorescent particles can be decreased or prevented by use of radical scavengers termed quenchers, oxygen depleting systems in solutions that contain the fluorescent biological reagent conjugates, chemical linkages in conjugates to minimize the effects of radicals, or combinations thereof.
Another embodiment of this invention comprises a system which consumes dissolved oxygen. For example, a system of this embodiment comprises catalase, glucose oxidase, and glucose, as discussed in greater detail herein.
In a preferred embodiment, solutions containing chemically oxidizable compounds that are associated with or conjugated to fluorescent molecules, also contain singlet oxygen and/or free radical quenchers to minimize or prevent oxidation of the chemical species. Oxidizable compounds include those that are capable of being converted from one oxidation state to a higher one. In general, most oxidations involve a gain of oxygen and/or a loss of hydrogen.
In another preferred embodiment, solutions containing biological reagents that are associated with or conjugated to fluorescent molecules, also contain singlet oxygen and/or free radical quenchers to minimize or prevent inactivation of the biological reagent. Biological reagents include, peptides and proteins containing oxidizable amino acids or pendant molecules, and nucleic acids.
In another preferred embodiment, solutions containing antibodies that are associated with or conjugated to fluorescent molecules or particles also contain singlet oxygen and/or free radical quenchers to minimize or prevent the ability of the antibody to bind its antigen. Antibodies include, polyclorial and monoclonal antibodies and binding fragments generated by recombinant protein synthesis or other techniques.
In yet another preferred embodiment, solutions containing nucleic acids that are associated with or conjugated to fluorescent molecules, also contain singlet oxygen and/or free radical quenchers to minimize or prevent the inactivation or slow the rate of hybridization of the nucleic acid to a complementary nucleic acid strand.
Compounds that can be utilized as quenchers of singlet oxygen include, but are not limited to those listed in Table 1. Compounds that can be utilized as quenchers of free radicals include, but are not limited to, those listed in Table 2.
A further aspect of the invention comprises use of heterofunctional reagents, such as heterobifunctional reagents, which have long molecular distances. The linkages produced thereby were used in fluorescent conjugates to associate a fluorescent molecule with a biological reagent. Accordingly, the biological reagent was at a maximized distance from the source of radical production. Maximizing the distance of the biological reagent from the source of the radical producer increased the time necessary for diffusion of the radical, and therefore increased the decay of the radical before it reached and then damaged the biological reagent. Such cross-linking reagents include but are not limited to: heterofunctional PEGs comprised of N-hydroxysuccinidyl (NHS) esters of poly (oxyethylene) derivatives containing a maleimide or vinyl sulfone group or similar electrophilic moiety; long chain forms of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amido-caproate), or of succinimidyl 3-(2-pyridyldithio)propionate (SPDP), such as succinimidyl-6-[3-(2-pyridyldithio)propionamido] hexanoate; and heterofunctional peptide derivatives.
An alternative embodiment of this invention utilized a protein substrate which was juxtaposed between a fluorescent molecule and a biological reagent. The protein substrate functioned to interact with the reactive species, such that the reactive species was quenched before it moved into the sphere of the biological reagent involved in that assay. This led to increased stability of the assay reagents over time. An example of this embodiment is set forth as Example 7.
Another embodiment of this invention incorporated water insoluble quenchers into a fluorescent particle conjugate. Such quenchers include but are not limited to quenchers of singlet oxygen, free radicals and excited triplet states; examples of these are given in Tables 1 and 2. A preferred quencher is the carotenoid xcex2-carotene, which can quench singlet oxygen, free radicals and excited triplet states; most other quenchers listed in Tables 1 and 2 possess the identified quenching activities. The water insoluble quenchers in physical contact with the dye molecules interacted efficiently with the reactive species, because they were closer to the source of production of the reactive species than the biological reagent involved in the assay. An example of this embodiment is set forth as Example 8.
Another embodiment of this invention comprised a combination of water soluble quenchers and emulsions of water insoluble quenchers, including but not limited to detergent micelles and liposomes, with a solution that contained the fluorescent conjugate or fluorescent particle conjugate. Examples of this embodiment are set forth as Examples 5 and 6.
Another embodiment of this invention comprised a combination of water insoluble quenchers incorporated into a fluorescent particle conjugated to a biological reagent, in combination with a system which consumes dissolved oxygen present in the solution that contains the conjugate.
Another embodiment of this invention comprised a combination of water soluble quenchers or emulsions of water insoluble quenchers in combination with a system which consumes dissolved oxygen in a solution that contains a fluorescent conjugate.
Another embodiment of this invention comprises a combination of both water insoluble quenchers incorporated into the fluorescent conjugate particle, water soluble quenchers or emulsions of water insoluble quenchers in the solution that contains the conjugate, together with a system which consumes dissolved oxygen in the solution that contains the fluorescent conjugate.